Evaluation and deployment of isotype-specific salivary antibody assays for detecting previous SARS-CoV-2 infection in children and adults.

BACKGROUND: Saliva is easily obtainable non-invasively and potentially suitable for detecting both current and previous SARS-CoV-2 infection, but there is limited evidence on the utility of salivary antibody testing for community surveillance. METHODS: We established 6 ELISAs detecting IgA and IgG antibodies to whole SARS-CoV-2 spike protein, to its receptor binding domain region and to nucleocapsid protein in saliva. We evaluated diagnostic performance, and using paired saliva and serum samples, correlated mucosal and systemic antibody responses. The best-performing assays were field-tested in 20 household outbreaks. RESULTS: We demonstrate in test accuracy (N = 320), spike IgG (ROC AUC: 95.0%, 92.8-97.3%) and spike IgA (ROC AUC: 89.9%, 86.5-93.2%) assays to discriminate best between pre-pandemic and post COVID-19 saliva samples. Specificity was 100% in younger age groups (0-19 years) for spike IgA and IgG. However, sensitivity was low for the best-performing assay (spike IgG: 50.6%, 39.8-61.4%). Using machine learning, diagnostic performance was improved when a combination of tests was used. As expected, salivary IgA was poorly correlated with serum, indicating an oral mucosal response whereas salivary IgG responses were predictive of those in serum. When deployed to household outbreaks, antibody responses were heterogeneous but remained a reliable indicator of recent infection. Intriguingly, unvaccinated children without confirmed infection showed evidence of exposure almost exclusively through specific IgA responses. CONCLUSIONS: Through robust standardisation, evaluation and field-testing, this work provides a platform for further studies investigating SARS-CoV-2 transmission and mucosal immunity with the potential for expanding salivo-surveillance to other respiratory infections in hard-to-reach settings.

If a person has been previously infected with SARS-CoV-2 they will produce specific proteins, called antibodies. These are present in the saliva and blood. Saliva is easier to obtain than blood, so we developed and evaluated six tests that detect SARS-CoV-2 antibodies in saliva in children and adults. Some tests detected antibodies to a particular protein made by SARS-CoV-2 called the spike protein, and these tests worked best. The most accurate results were obtained by using a combination of tests. Similar tests could also be developed to detect other respiratory infections which will enable easier identification of infected individuals.

Adenosine 2A receptor and TIM3 suppress cytolytic killing of tumor cells via cytoskeletal polarization.

Tumors generate an immune-suppressive environment that prevents effective killing of tumor cells by CD8+ cytotoxic T cells (CTL). It remains largely unclear upon which cell type and at which stage of the anti-tumor response mediators of suppression act. We have combined an in vivo tumor model with a matching in vitro reconstruction of the tumor microenvironment based on tumor spheroids to identify suppressors of anti-tumor immunity that directly act on interaction between CTL and tumor cells and to determine mechanisms of action. An adenosine 2A receptor antagonist, as enhanced by blockade of TIM3, slowed tumor growth in vivo. Engagement of the adenosine 2A receptor and TIM3 reduced tumor cell killing in spheroids, impaired CTL cytoskeletal polarization ex vivo and in vitro and inhibited CTL infiltration into tumors and spheroids. With this role in CTL killing, blocking A2AR and TIM3 may complement therapies that enhance T cell priming, e.g. anti-PD-1 and anti-CTLA-4.

In situ characterization of stem cells-like biomarkers in meningiomas.

BACKGROUND: Meningioma cancer stem cells (MCSCs) contribute to tumor aggressiveness and drug resistance. Successful therapies developed for inoperable, recurrent, or metastatic tumors must target these cells and restrict their contribution to tumor progression. Unfortunately, the identity of MCSCs remains elusive, and MSCSs' in situ spatial distribution, heterogeneity, and relationship with tumor grade, remain unclear. METHODS: Seven tumors classified as grade II or grade III, including one case of metastatic grade III, and eight grade I meningioma tumors, were analyzed for combinations of ten stem cell (SC)-related markers using immunofluorescence of consecutive sections. The correlation of expression for all markers were investigated. Three dimensional spatial distribution of markers were qualitatively analyzed using a grid, designed as a repository of information for positive staining. All statistical analyses were completed using Statistical Analysis Software Package. RESULTS: The patterns of expression for SC-related markers were determined in the context of two dimensional distribution and cellular features. All markers could be detected in all tumors, however, Frizzled 9 and GFAP had differential expression in grade II/III compared with grade I meningioma tissues. Correlation analysis showed significant relationships between the expression of GFAP and CD133 as well as SSEA4 and Vimentin. Data from three dimensional analysis showed a complex distribution of SC markers, with increased gene hetero-expression being associated with grade II/III tumors. Sub regions that showed multiple co-staining of markers including CD133, Frizzled 9, GFAP, Vimentin, and SSEA4, but not necessarily the proliferation marker Ki67, were highly associated with grade II/III meningiomas. CONCLUSION: The distribution and level of expression of CSCs markers in meningiomas are variable and show hetero-expression patterns that have a complex spatial nature, particularly in grade II/III meningiomas. Thus, results strongly support the notion of heterogeneous populations of CSCs, even in grade I meningiomas, and call for the use of multiple markers for the accurate identification of individual CSC subgroups. Such identification will lead to practical clinical diagnostic protocols that can quantitate CSCs, predict tumor recurrence, assist in guiding treatment selection for inoperable tumors, and improve follow up of therapy.

Mononuclear phagocytes contribute to intestinal invasion and dissemination of Yersinia enterocolitica.

Enteropathogenic Yersinia enterocolitica (Ye) enters the host via contaminated food. After colonisation of the small intestine Ye invades the Peyer's patches (PPs) via M cells and disseminates to the mesenteric lymph nodes (MLNs), spleen and liver. Whether Ye uses other invasion routes and which pathogenicity factors are required remains elusive. Oral infection of lymphotoxin-ß-receptor deficient mice lacking PPs and MLNs with Ye revealed similar bacterial load in the spleen 1h post infection as wild-type mice, demonstrating a PP-independent dissemination route for Ye. Immunohistological analysis of the small intestine revealed Ye in close contact with mononuclear phagocytes (MPs), specifically CX3CR1(+) monocyte-derived cells (MCs) as well as CD103(+) dendritic cells (DCs). This finding was confirmed by flow cytometry and imaging flow cytometry analysis of lamina propria (LP) leukocytes showing CD103(+) DCs and MCs with intracellular Ye. Uptake of Ye by LP CD103(+) DCs and MCs was dependent on the pathogenicity factor invasin, whereas the adhesin YadA was dispensable as demonstrated by Ye deletion mutants. Furthermore, Ye were found exclusively associated with CD103(+) DCs in the MLNs from wild-type mice, but not from CCR7(-/-) mice, demonstrating a CCR7 dependent transport of Ye by CD103(+) DCs from LP to the MLNs. In contrast, dissemination of Ye to the spleen was dependent on MCs as significantly less Ye could be recovered from the spleen of CX3CR1(GFP/GFP) mice compared to wild-type mice. Altogether, MCs and CD103(+) DCs contribute to immediate invasion and dissemination of Ye. This together with data from other bacteria suggests MPs as general pathogenic entry site in the intestine.